The detection of phosphorylated proteins holds great promise in delineating many of the signal transduction pathways that occur in normal and diseased cells. The possibility of studying the phospho-proteome as an indicator of the drug efficacy is gaining popularity in the pharmaceutical industry, in part due to the disappointing results obtained using standard genomic approaches.
Classic analysis of cell signaling pathways uses cell lines incubated in the presence and/or absence of an activator or inhibitor. Proteins are then extracted and analyzed by Western blot using one or more anti-phospho-protein antibodies. Unfortunately, this approach is time consuming, expensive and not amenable to high throughput screening.
Flow cytometry has advantages over Western blot, in that specific subsets of cells can be targeted due to reproducible light scatter profiles obtained when cells are interrogated by laser light combined with various fluorescent antibodies. Further, flow cytometry can be used to detect intracellular phospho-epitopes. In comparing cytometric detection to Western blots, several other advantages surface, including, but not limited to: 1) a large dynamic range of data collection (typically 10,000 fold), 2) rapid protocols that take 2 hours, not 2 days, 3) simultaneous analysis of multiple epitopes in the same cell, and 4) the possibility of quantitation in a single cell.
The literature describing cytometric detection of phospho-epitopes is limited to two seminal papers. In the first paper, Chow, et al. (1) described a technique for whole blood or isolated cells that detected inhibition in the MAP kinase pathway using phospho-specific antibodies to MEK/ERK. The authors list the destruction of surface epitopes and poor light scatter resolution as detrimental to the analysis. In a more “proof of principle” sense, Perez, et al. (2) demonstrated the detection of multiple epitopes using multiple fluorochromes on isolated lymphocytes and cell lines.
In both papers, the authors allude to the fact that if a fixation technique could be devised that maintained surface epitopes together with resolvable light scatter, the use of cytometry would be more broadly applicable. However, to date no satisfactory fixative has been found that maintains both surface and intracellular epitopes, light scatter properties of the cell, and DNA profiles.
Current fixatives revolve primarily around alcohol and formaldehyde/paraformaldehyde (3). Alcohols dehydrate the cell allowing immediate internal access, but are detrimental to most surface epitopes and cause the cells to aggregate. The crosslinkage of proteins is the attractive feature of paraformaldehyde fixatives. However, this feature denies access to proteins in their native state and is detrimental to DNA dyes.
What is needed in the field is a fixative technique that 1) maintains easily resolvable light scatter patterns, 2) preserves surface epitopes, 3) preserves intracellular epitopes, and 4) allows DNA content analysis if so desired. An added benefit would accrue if the fixative could be used on whole blood or bone marrow due to its ability to lyse mature red blood cells (RBC).
The fixative described by Connelly (4a) is the best single step fixative and permeation agent discovered to date (see e.g., reference (8) stating that “Best results were obtained using a commercial reagent Ortho PermeaFix (OPF) for flow cytometry”). It is called Ortho PERMEAFIX™, although that product has been replaced with a new product called PERMIFLOW™ (INVIRION, INC.™ MI). OPF and its variants are well described in U.S. Pat. No. 5,422,277 and U.S. Pat. No. 5,597,688. Preferred fixatives comprised 0.756%-0.85% formaldehyde, 25.4-30 mM DNBS, 6.9-6.92% DMSO and 0.086-0.095% TWEEN™ 20 detergent, although many variations are described.
OPF fixation is asserted to have “maintained the morphology of lymphoid cells with minimal cellular distortion and scatter changes, and only slightly modified cell surface immunoreactivity.” (4a). In fact, Connelly has successfully applied this fixative to the detection of both surface and intracellular antigens (4b, 5), and a particular benefit is that an additional red blood cell (RBC) lysing reagent was not required because RBC lysis occurred upon resuspension of OPF-treated whole blood samples in isotonic solution. Others have shown that unlike most other fixatives, OPF is also compatible with DNA staining (6).
The inventors of OPF specifically teach that “the temperatures maintained during such [fixation] incubation are generally 0° C. to about 37° C., with room temperature preferable” (5). In contrast to the patent, however, a scientific publication by the Connelly group (4a) states that morphology is improved at 4° C. over a room temperature fixation. In fact, most cell preparation techniques for flow cytometry require fixation on ice or at most room temperature, because the lower temperature is believed to be required to maintain the cell's metabolic state until fixation, and to maintain cell morphology during and after fixation.
Unfortunately, many antigens cannot be detected after room temperature fixation (see, e.g., (7) and the results described herein). Thus, an improved fixative method is needed in the art.